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Fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate in the glycolytic pathway. FBPAs from archaeal organisms have recently been identified and characterized as a divergent family of proteins. Here, we report the first crystal structure of an archaeal FBPA at 1.9-Å resolution. The structure of this 280-kDa protein complex was determined using single wavelength anomalous dispersion followed by 10-fold non-crystallographic symmetry averaging and refined to an R-factor of 14.9% (R free 17.9%). The protein forms a dimer of pentamers, consisting of subunits adopting the ubiquitous (␤␣) 8 barrel fold. Additionally, a crystal structure of the archaeal FBPA covalently bound to dihydroxyacetone phosphate was solved at 2.1-Å resolution. Comparison of the active site residues with those of classical FBPAs, which share no significant sequence identity but display the same overall fold, reveals a common ancestry between these two families of FBPAs. Structural comparisons, furthermore, establish an evolutionary link to the triosephosphate isomerases, a superfamily hitherto considered independent from the superfamily of aldolases.The fructose-1,6-bisphosphate aldolase (FBPA, 1 EC 4.1.2.13) fulfills an important function in the reversible Embden-Meyerhof-Parnas pathway and the Calvin cycle. The enzyme catalyzes the reversible aldol condensation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Two different types of FBPA, termed class I and II (FBPA I and FBPA II), with different enzymatic mechanisms have so far been discovered. Whereas class I enzymes utilize the ⑀-amino group of the active site lysine to form a Schiff base with the substrate, the FBPAs II use divalent metal ions to stabilize the intermediate (1). The classical FBPA I is mainly found in eukaryotes (e.g. animals, higher plants, protozoa, algae, and yeast) and is only identified in a few bacteria. FBPA II is in contrast mainly used by bacteria (2). Although both classes of enzymes adopt the (␤␣) 8 -(TIM)-barrel fold, their insignificant overall sequence similarity casts doubts on whether they share a common ancestor. However, more recent analyses based on sequence, structure, and functional information suggest that both FBPA classes share a common evolutionary origin (3, 4).The recently identified archaeal FBPA (FBPA IA) represents a third type of aldolase. Archaeal organisms appear to rely solely on the archaeal class I enzyme for FBPA activity (5). Although these enzymes use the Schiff base mechanism, they do not show any significant overall sequence similarity to classical FBPA I. Some bacterial genomes contain FBPA IA homologs (6) in addition to FBPA II, the typical bacterial FBPA (e.g. Escherichia coli) (2). So far no eukaryotic homologs of the archaeal class I enzymes are known.Biochemical analyses of archaeal FBPA from Thermoproteus tenax and Pyrococcus furiosus reveal that this enzyme, like the classical FBPA I, catalyzes th...

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The triosephosphate isomerase of the hyperthermophilic crenarchaeum Thermoproteus tenax (TtxTIM) represents a homomeric tetramer. Unlike the triosephosphate isomerases of other hyperthermophiles, however, the association of the TtxTIM tetramers is looser, allowing a reversible dissociation into inactive dimers. The dimer/tetramer equilibrium of TtxTIM is shifted to the tetrameric state through a specific interaction with glycerol-1-phosphate dehydrogenase of T. tenax, suggesting that higher oligomerization of the TtxTIM serves functional rather than stabilizing purposes.

Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen.